Made available in DSpace on 2016-08-15T20:27:59Z (GMT). No. of bitstreams: 1 IvanaCNGL_TESE.pdf: 14299009 bytes, checksum: 5bf2d46500f8d157d958429f1dc7dad5 (MD5) Previous issue date: 2015-04-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Gossypol is a phenolic compound produced by pigment glands in cotton stems, leaves, seeds and flower buds (Gossypium spp). Cottonseed meal is a by-product of cotton that is used for animal feeding because it is rich in oil and proteins. However, gossypol toxicity limits cottonseed use in animal feed. High concentrations of free gossypol may be responsible for acute clinical signs of gossypol poisoning, the most common toxic effect is a reduction in reproduction of males and females. The objective of this study was to evaluate the effects of gossypol in the estrous cycle, folliculogenesis in vivo and in vitro and in developing embryos in vitro rodent and chickens. For the first experiment were used 24 Wistar rats divided into two groups: a control group (saline injection, subcutaneously) and the other treated with gossypol (25 mg/kg/day subcutaneously) for 15 days. The rats were euthanized after the last day of treatment for blood and fragments of ovaries, uterus and thyroid. Blood was collected for hormone analysis by ELISA and the ovaries were collected for histological study for qualitative and quantitative evaluation of ovarian follicles. For the second experiment, it took 32 ovaries of female rats and mice and four chickens. The ovaries of rats and mice, chickens and fragments ovary cells were grown in 24-well culture plates containing 1 ml of culture medium specific. We tested four concentrations of gossypol: 0 (control), 5, 10 and 20 µg / ml. All plates were incubated at 39 ° C and 5% CO2 for 24 hours and seven days. After incubation, the ovaries were fixed and processed for histological analysis. For the third experiment, nulliparous female Balb-C mice were superovulated and then allocated to males. Next day, harvest embryos was performed in 2-8 cell stage. Sequential washes the tubes were performed to remove the embryos on modified HTF. Lots 5 - 8 are selected embryos collected and transferred to new culture HTF medium without HEPES supplemented with 10% FBS. Subsequently they were collected and deposited into microdroplets (30 µ L) medium supplemented in 35x10 mm plates. The embryos were cultured in medium without addition of gossypol (control group) or with concentrations of 5, 10 and 20 µg / ml of gossypol added to each drop. These droplets were immersed in mineral oil and grown in a glasshouse with 5% CO2 at 37 ° C for 24 hours. After the cultivation, the embryos were stained for the detection of ruptured membranes and cells in cell death. It can be seen that gossypol promotes degeneration of ovarian follicles and embryolethality and this toxicity occurs by direct action of gossypol and not a product of its biotransformation / Gossipol é um composto fenólico produzido pelas glândulas de pigmento presente nas raízes, caules, folhas, sementes e botões de flores do algodão (Gossypium spp). O farelo e a torta de algodão são subprodutos do algodão utilizados para a alimentação animal, pois são ricos em óleo e proteínas. No entanto, o gossipol limita a utilização do algodão na alimentação animal, pois altas concentrações desta substância podem ser responsáveis por sinais clínicos agudos de intoxicação, sendo o efeito tóxico mais comum é a redução na reprodução de machos e fêmeas. O objetivo deste estudo foi avaliar os efeitos do gossipol no ciclo estral, na foliculogênese in vivo e in vitro e no desenvolvimento de embriões in vitro de roedores e galinhas. Para o primeiro experimento, foram utilizadas 24 ratas Wistar, distribuídas em dois grupos iguais: um grupo controle (injeção de solução salina, por via subcutânea) e o outro tratado com gossipol (25 mg/kg/dia, por via subcutânea) durante 15 dias. As ratas sofreram eutanásia após o último dia do tratamento para coleta de sangue e de fragmentos de ovários, útero e tireoide. Foi coletado sangue para análise hormonal por ELISA e os ovários foram coletados para estudo histológico para avaliação qualitativa e quantitativa dos folículos ovarianos. Para o segundo experimento, foram necessários 32 ovários de ratas e camundongas e quatro de galinhas. Os ovários das ratas e de camundongas, e fragmentos dos ovários de galinhas foram cultivadas em placas de 24 poços de cultura de células contendo 1 ml de meio de cultura específico. Foram testadas quatro concentrações de gossipol: 0 (controle), 5, 10 e 20 ug/mL. Todas as placas foram incubadas a 39 °C e 5% de CO2 durante 24 horas e sete dias. Após a incubação, os ovários foram fixados e processados para análise histológica. Para o terceiro experimento, fêmeas nulíparas de camundongos Balb-C foram superovuladas e em seguida, alocadas com machos. No dia seguinte, foi realizada a colheita de embriões na fase de 2-8 células. Foram realizadas lavagens sequenciais das tubas para remoção dos embriões com meio HTF modificado. Lotes de 5 8 embriões coletados e selecionados foram transferidos para novo meio de cultivo HTF sem HEPES suplementado com 10% de SFB. Posteriormente foram recolhidos e depositados em microgotas (30 µl) de meio suplementado em placas de 35x10 mm. Os embriões foram cultivados em meio sem a adição de gossipol (grupo controle) ou com concentrações de 5, 10 e 20 µg/ml de gossipol, adicionadas em cada gota. Estas gotas foram imersas em óleo mineral e cultivadas em estufa com 5% de CO2 a 37 °C, por 24 horas. Após o cultivo, os embriões foram corados para a detecção de membranas rompidas e de células em processo de morte celular. Pode-se observar que o gossipol promove degeneração de folículos ovarianos e embrioletalidade, e esta toxicidade ocorre por meio de ação direta do gossipol e não por algum produto de sua biotransformação

The giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879), is gaining popularity as a key aquaculture species; global production currently exceed 220,000 tonnes, however, industry expansion is limited by high operational costs, with the feed accounting for between 40 to 60% of these. Attention, therefore, has been devoted to increasing the inclusion of plant proteins into the formulation of aqua feeds as a consequence of the limited, unpredictable supply and increasing price of fishmeal. The concomitant introductions of anti-nutritional factors (ANFs), such as phytic acid (PA) with the plant protein fraction, however, are major impediments in the efforts toward the increased use of plant protein ingredients in aqua feeds. Phytic acid is an anti-nutrient that can curtail the development of this as PA has been reported to suppress growth impairing proper nutrient intake, diminishing the availability of minerals, and causing damage to the body tissues and organs which can result in mortality. Although the anti-nutritive effects of PA have been studied extensively in terrestrial agriculture farm species, as well as in a variety of fish species, there is almost no information regarding the effects of PA in crustaceans, including the freshwater prawn, M. rosenbergii. The aims of this present thesis were, therefore, to gain a greater understanding of dietary PA and microbial phytase and their effect on growth performance, feed utilisation, nutrient utilisation and digestibility, mineral availability and whole body proximate composition in juvenile M. rosenbergii. Specifically, the first major experiment set out to investigate the effect of including increasing amounts of PA in the diets presented to M. rosenbergii on growth. The dose-response relationships between PA and growth performance, feed utilisation, nutrient digestibility and utilisation and whole proximate composition were investigated. Four replicate groups of M. rosenbergii with a mean initial carapace length of 6.03 ± 0.30 mm and mean initial weight of 0.29 ± 0.02 g were fed graded levels of PA for 140 days. The basal diet, to which different levels of PA were added to obtain 0.26 (control), 6.48, 11.28, 16.53, 21.45 and 26.16 g PA kg-1, contained fishmeal, soy protein concentrate, wheat meal and corn starch. The results indicated that growth performance, feed utilisation and survival did not differ significantly between the groups receiving the different inclusions of PA within their diets. The apparent protein, lipid and energy utilisations responded negatively, decreasing significantly (p<0.05) with an increasing inclusion of PA, particularly within the groups of prawns fed the diet with the highest inclusions of PA, i.e. the 21.45–26.16 g PA kg-1 diets. The digestibility of protein and lipid were also reduced as the inclusion of PA increased. The whole body composition of protein (p<0.04), lipid (p<0.01) and gross energy (p<0.05) decreased significantly with an increasing supplementation of PA, while the ash content significantly increased (p<0.01), most notably in the groups of prawns receiving the highest levels of dietary PA. The second major experimental trial investigated the effect of microbial phytase on the growth of juvenile M. rosenbergii, when fed diets supplemented with various doses for a period of 80 days. The study set out to improve the growth performance, feed utilisation, nutrient digestibility and utilisation and body composition of M. rosenbergii when fed diets high in plant protein ingredients. To investigate this, four plant protein based diets, which included soybean meal, wheat gluten and wheat meal, were formulated and supplemented with microbial phytase at levels of 0, 500, 1000 and 2000 FTU kg-1 (one phytase unit per kg) and fed to sixty juvenile M. rosenbergii (mean initial carapace length of 8.51 ± 0.52 mm; mean initial weight of 0.40 ± 0.07 g) for 80 days. High levels of plant protein in the diets supplemented with 0–2000 FTU kg-1 did not result in any negative effect on growth performance, feed utilisation nor on the survival of M. rosenbergii. Noticeable moderate growth improvements in line with increasing microbial phytase supplement levels were observed and the highest growth performance was seen in the group fed 2000 FTU kg-1. Supplementation of the diets with 500–2000 FTU kg-1 were found to affect the nutrient utilisation, resulting in a significant (p<0.05) increase in the protein and lipid utilisation when compared to the prawns analysed from the control group. In addition to this latter finding, an increasing supplement of phytase in the diet also resulted in an increase in the dry matter fraction as well as improvements in the digestibility of protein and lipid. There were, however, no significant differences in moisture, protein, lipid, gross energy and the ash content of the whole body of the M. rosenbergii among the groups. This thesis, in a third major trial, explored the impact of other potential ANFs associated to PA, the binding effect of PA with mineral. The effects of graded levels of PA (i.e. 0.26–control, 6.48, 11.28, 16.53, 21.45 and 26.16 g PA kg-1) on the moult frequency and mineral availability in juvenile M. rosenbergii fed over a period of 140 days were determined. The levels of PA assessed in this feed trial had no major detrimental effects on moult frequency. Negative effects (p<0.05) of high PA levels (i.e. 21.45–26.16 g PA kg-1), however, were found on the whole body P concentration. An inverse trend was recorded for the Ca content in the whole body (p<0.005) and carapace (p<0.004) with increasing PA inclusion. These results are consistent with findings for marine shrimp species such as Marsupenaeus japonicus and Litopenaeus vannamei. The graded inclusion of PA in the experimental diets also resulted in a significant reduced (p<0.03) P content in the carapace. Significant changes (p<0.05) were observed in the carapace Zn, Cu, K and Na compositions, particularly in the prawns fed the diet containing 11.28 g PA kg-1, which suggests that the specific minerals were either selectively utilised or retained in the carapace. Accordingly, this thesis investigates the potential of adding dietary supplements of microbial phytase in order to improve mineral availability as proven in several fish species. To explore this, four experimental diets were formulated - three incorporating different levels of microbial phytase (i.e. 0 FTU kg-1, 1000 FTU kg-1 and 2000 FTU kg-1) and a fourth, a control. An aliquot of 15 g PA kg-1 was also added to each treatment. The mineral premix was omitted from the test group diets except the control diet was identical to the 0 FTU kg-1 diet but included a mineral premix and an aliquot of 8 g kg-1 monosodium phosphate which replaced an equal amount of wheat meal fraction. The trial found no significant differences in growth, feed utilisation and moult frequency with the microbial phytase level within the diet, however, survival was compromised. The proximate composition of the prawn whole body was in most cases unaffected by the level of phytase. Supplementation of the diets with microbial phytase did, however, result in significant higher (p<0.05) concentrations of minerals including: 1) Ca, Mg, K and Na in the whole body; 2) Ca and Zn in muscle tissue; and, 3) Ca and Mg in the carapace. The Zn content of the carapace, however, was negatively affected by the inclusion of microbial phytase suggesting the necessity of this mineral within the diet of M. rosenbergii. This thesis contributes to current understanding surrounding the inclusion of dietary PA and the benefits of microbial phytase within the experimental diets consumed by juvenile M. rosenbergii. The knowledge gained from this work provides a means to optimise the use of plant protein ingredients and with the potential to decrease the dependability of fishmeal without compromising M. rosenbergii production and profitability, thus ultimately promoting the sustainable expansion of M. rosenbergii aquaculture.